Received August 30, 2016; Revision received October 12, 2016
Long noncoding RNAs (lncRNAs) have been recently regarded as systemic
regulators in multiple biological processes including tumorigenesis. In
this study, we report an ultra-highly expressed lncRNA, lnc-Sox5, in
tongue tumor tissues. The results imply that lnc-Sox5 may play vital
role in tongue carcinoma progression. We observed that the growth of
Tca8113 cells was suppressed by lnc-Sox5 downregulation. Additionally,
lnc-Sox5 knockdown simultaneously increased Tca8113 cell apoptosis, but
the cell cycle was arrested. RNA immunoprecipitation suggested that HuR
directly bound to and stabilized lnc-Sox5 RNA. Consistently, HuR
knockdown reduced the level of lnc-Sox5 in Tca8113 cells. However,
overexpression of HuR induced more lnc-Sox5 in Tca8113 cells. Both
lnc-Sox5 knockdown and HuR knockdown suppressed Tca8113 cell
tumorigenesis in xenograft models. These results suggest that lnc-Sox5,
which was stabilized by HuR, could regulate carcinogenesis of tongue
cancer and may serve as a predicted target for tongue carcinoma
therapies.
KEY WORDS: long non-coding RNA, lnc-Sox5, tongue carcinoma, cell
cycle, apoptosis, HuR, RNA immunoprecipitation

DOI: 10.1134/S0006297917040046

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common
cancer worldwide including tumors of oropharynx, oral cavity,
hypopharynx, and larynx [1]. Tongue squamous cell
carcinoma (TSCC) is the most common type of oral tumors and is
characterized by high metastatic rate of lymph nodes [2]. Multiple factors including smoking, alcohol
consumption [3, 4], and human
papilloma virus (HPV) infection [5, 6] have been demonstrated to increase the risk of
tongue squamous cell carcinoma. Despite great advances achieved in TSCC
therapy, the mortality rate remains high. Therefore, it is urgent to
clarify the mechanism and to identify new factors in tongue
carcinogenesis.

Long noncoding RNAs (lncRNAs) are generally deﬁned as
un-translational transcripts composed of more than 200 nucleotides [7], and they are associated with many important
cellular processes and pathogenesis. Numerous lncRNAs are involved in
tongue carcinogenesis and metastasis, such as MALAT1 [8], HOTTIP [9], MEG3 [10], and UCA1 [11]. Despite many
lncRNAs being expressed at higher or lower level in tongue tumor
tissues than them in adjacent tissues, their role and mechanism in
tumorigenesis are not clearly elucidated.

In this study, we identified an ultra-highly expressing lncRNA lnc-Sox5
in tongue squamous cell carcinoma. To investigate whether this
abnormally expressing lncRNA regulates TSCC progression, we first
specifically suppressed lnc-Sox5 in Tca8113 cells by siRNA
transfection. Interestingly, the growth of Tca8113 cells was
dramatically suppressed by lnc-Sox5 knockdown. Additionally, the
apoptosis of Tca8113 cells was simultaneously enhanced by lnc-Sox5
knockdown. Furthermore, the cell cycle was also arrested at G1 to S
phase by lnc-Sox5 suppression. To test whether lnc-Sox5 affected tongue
cancer metastasis, transwell assays were performed for examining the
invasion and migration of Tca8113 cells. These results suggested that
both migration and invasion of Tca8113 cells were suppressed by
lnc-Sox5 knockdown. A xenograft tumor model suggested that lnc-Sox5
promoted Tca8113 cell carcinogenesis. Moreover, with bioinformatics
analysis we observed that there are several HuR binding sites in
lnc-Sox5 mRNA. RNA immunoprecipitation (RIP) assay indicated that HuR
directly bound to lnc-Sox5 mRNA. Taken together, we report here a new
lncRNA, lnc-Sox5, to promote tumorigenesis of tongue squamous cell
carcinoma.

MATERIALS AND METHODS

Cells and tissues. Human cell lines HEK-293, HeLa, and U266 were
obtained from the Cell Bank of the Chinese Academy of Sciences
(Shanghai, China) and Tb3.1, Tca8113, SCC-4, CTSC-3, and Cal27 cells
were obtained from the Department of Oncology (Second Affiliated
Hospital of Qingdao University). HEK-293 and HeLa cells were cultured
in Dulbecco’s modified Eagle medium (DMEM) and U266, Tb3.1,
Tca8113, SCC-4, CTSC-3, and Cal27 cells were cultured in RPMI-1640
medium supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen,
USA) at 37°C in a humidified atmosphere containing 5%
CO2. All pairs of TSCC tissues and adjacent tongue tissues
were collected from patients who underwent tumor resection at the
Second Affiliated Hospital of Qingdao University. All samples were
pathologically confirmed, and these patients did not receive any
anticancer treatment before surgery. All tissues were acquired with
approval of patients, and the protocol was reviewed and approved by the
Ethics Committee of the Second Affiliated Hospital of Qingdao
University. Fresh tissue samples were snap-frozen in liquid nitrogen
and stored at –80°C until RNA or protein extraction.

Plasmids and retrovirus packaging. DNA fragments containing HuR
complement sequence were inserted into pMSCV-PIG plasmid. The lnc-Sox5
and HuR siRNA were inserted into pMDH-PGK-EGFP2.0 (pMDH) vector. The
sequence of siRNA to lnc-Sox5 and HuR are listed in the table.
Retroviral supernatant was generated using standard procedures after
calcium phosphate cotransfection of pMDH-siRNA and pAmpho viral
packaging plasmid into HEK293T cells. pMDH-siRNA table expression cell
lines were generated by virus infection and selected using flow
cytometry (FACSAria III; BD Biosciences, USA).

Sequences of primers and siRNAs used in this study

qRT-PCR. Total RNA was purified from TSCC tissues or Tca8113
cells following the manufacturer’s instructions. To detect the
expression of lnc-Sox5, cDNA was synthesized using a Reverse
Transcription Kit (Takara, China). qRT-PCR was performed using SYBR
Green Master Mix (Roche, Switzerland). All primers are listed in the
table. The mRNA levels were determined by the comparative Ct method
after normalization to β-actin.

Transwell assays. Boyden chambers were used for cell migration
assays. Tca8113 cells (2·105 cells/well) were placed
in the upper transwell chambers (8 µm pore size) 24 h after
siRNA transfection. The lower chambers were filled with 0.5 ml
RPMI-1640 containing 5% FBS. Cells were incubated for 24 h at
37°C in 5% CO2. Nonmigratory cells were carefully
scraped with a cotton swab. Cells remaining on the bottom surface were
counted after staining with crystal violet. The number of cells in five
random fields was counted for cell migration analysis. The same Boyden
chambers were used for cell invasion assay. The upper surface of the
filter was coated with 20 µl Matrigel (0.3 mg/ml; BD
Biosciences). Cells were incubated for 24 h at 37°C in 5%
CO2. Cells were counted as described above.

Cell proliferation assay. Tca8113 cells were seeded in 96-well
plates (4·103 cells per well) and transfected with
lnc-Sox5 siRNAs or mimics control and cultured at 37°C in an
incubator containing 5% CO2. After culturing for 24 h,
cells were tested for viability using the
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
assay. Briefly, cells were incubated with MTT at final concentration
0.5 mg/ml (Sigma, USA) for 4 h. The supernatant was
discarded, and the precipitated formazan was dissolved in dimethyl
sulfoxide. Absorbance was measured at 570 nm with a microplate
reader (Molecular Devices, USA).

Bioinformatics analysis. About 118,777 transcripts were
downloaded from LNCipedia (www.lncipedia.org). All these transcripts
containing AT-rich elements were selected as the candidates for HuR
target.

RNA immunoprecipitation (RIP). Tca8113 cells were collected and
washed with PBS, and the pellets were disintegrated in RIP buffer on
ice for 30 min and centrifuged at 10,000g for 5 min.
Anti-HuR antibodies (5 µg) were added to the supernatant and
incubated for 4 h at 4°C. Protein A beads (40 µl) were
added and incubated for another 2 h at 4°C. The beads were pelleted
at 1600g for 30 s, the supernatant was removed, and the
beads were resuspended in 500 ml of RIP buffer. The RIP wash was
repeated thrice, followed by one wash in PBS. Coprecipitated RNAs were
isolated by resuspending the beads in TRIzol RNA extraction reagent
(500 µl) according to the manufacturer’s instructions.
Purified RNA was subject to quantitative PCR for lnc-Sox5 determination
as described above.

Animal tumor model. Six-to-eight-week-old BALB/c (nu/nu) mice
were purchased from Shanghai SLAC Laboratory Animal Co (China). The
mice were maintained in a barrier facility at the Animal Center of
Chongqing Medical University. Stably expressing lnc-Sox5 siRNA or
scramble control cells were implanted subcutaneously (s.c.) into the
right flank of the mice. Tumor volume was measured three times per
week. All groups of mice were sacrificed, and tumors were weighted at
the endpoint of these experiments.

Statistical analysis. Comparison between groups was performed
using Student’s t-test and one-way ANOVA. SPSS 16.0 (SPSS,
USA) was used for all statistical analyses. Experiments were performed
at least three times. All results are described as means ±
standard error of mean (S.E.M); p values less than 0.05 were
considered as significant differences. All tests were done using Prism
6 software (Graphpad Prism 6).

RESULTS

Aberrant lnc-Sox5 expression in tongue squamous cell carcinoma and
tongue cancer cell lines. To explore abnormally expressing lncRNAs
in tongue squamous cell carcinoma, we examined the level of several
lncRNAs in TSCC tissues (data not shown). We observed that the
expression of lnc-Sox5 was significantly increased in TSCC. Therefore,
22 pairs of clinic TSCC tissues and matched adjacent normal tissues
were subjected to quantitative PCR (q-PCR) for lnc-Sox5 expression.
Interestingly, almost all matched samples showed significantly
increased level of lnc-Sox5 in tumor tissues compared with their normal
control, whereas only three samples showed downregulated or equivalent
lnc-Sox5 level in TSCC tissues (Fig. 1a). To
investigate whether lnc-Sox5 played a functional role in TSCC
progression, we subsequently detected the expression of lnc-Sox5 by
q-PCR in several TSCC cell lines, including Tca8113, Tb3.1, SCC-4,
CTSC-3, Cal27 and other cancer cell lines, such as 293T, HeLa, and U266
cells (Fig. 1b). Consistently, lnc-Sox5 was found
to be expressed at high level in tongue cancer cell lines. These
results suggested that the aberrant lnc-Sox5 might act in a potential
oncogenic role in TSCC progression.

Lnc-Sox5 impaired Tca8113 cell apoptosis and cell cycle in
vitro. To extensively investigate the oncogenic role of
lnc-Sox5 in TSCC, we selected Tca8113 cells for further experimental
analysis because lnc-Sox5 was found to be expressed at the highest
level in this cell line (Fig. 1b). First, we
specifically suppressed the expression of lnc-Sox5 by siRNA
transfection. As the result described, the first siRNA (siRNA1#) had
the best inhibitory efficacy in Tca8113 cells (Fig. 2a). MTT result showed that the cell growth was
significantly suppressed by lnc-Sox5 knockdown (Fig. 2b). Additionally, the apoptotic rate of Tca8113 cells
was increased by lnc-Sox5 knockdown (Fig. 2, c and
d). Flow cytometry showed that the cell cycle was also retarded by
lnc-Sox5 suppression (Fig. 3, a and b). These
results suggested that lnc-Sox5 promoted cell growth in Tca8113 cells.
To clearly understand the oncogenic role of lnc-Sox5 in tongue cancer,
the migration and invasion of Tca8113 cells in vitro were also
investigated. The invasion and migration of Tca8113 cells were analyzed
by transwell assays. Interestingly, both invasion and migration of
Tca8113 cells were decreased after lnc-Sox5 inhibition (Fig. 4, a and b). Taken together, these data suggested that
lnc-Sox5 acted as an oncogene in tongue cancer progression.

Fig. 3. Loss of lnc-Sox5 retarded Tca8113 cell cycle at G0/1
to S phase. a, b) Tca8113 cells were stained with PI as described
above, and the cell cycle was determined by flow cytometry after
lnc-Sox5 siRNA1#.

Lnc-Sox5 knockdown suppressed tongue tumor growth. To extensively
explore the oncogenic role of lnc-Sox5 in tongue cancer progression, we
subsequently generated a stably lnc-Sox5 knockdown Tca8113 cell line by
infecting cells with retrovirus expressing siRNA1. Furthermore, we used
this cell line to construct a xenograft mouse model, and the tumor
volume was detected every two days when the tumor volume reached 100
mm3. As expected, lnc-Sox5 knockdown significantly
suppressed the tumorigenesis of Tca8113 cells (Fig. 5a). Consistent to the tumor volume, tumor weight was
also significant reduced by lnc-Sox5 knockdown (Fig. 5b). According to these results, we conclude that
lnc-Sox5 plays an oncogene in tongue cancer progression.

Lnc-Sox5 expression is associated with and stabilized by HuR.
Lnc-Sox5 contributed to tongue cancer carcinoma, prompting us to
investigate the regulatory mechanism of controlling lnc-Sox5 expression
in tongue cancer cells. HuR is a RNA-binding protein that directly
binds to messenger RNA frequently containing AU- or U-rich sequence
elements (ARE). Thus, we first explored the sequence of lnc-Sox5 mRNA.
Surprisingly, there were many AU-rich sequences distributed in the full
length of lnc-Sox5 (http://www.lncipedia.org/db/transcript/lnc-SOX5).
Therefore, we subsequently detected the interaction between HuR and
lnc-Sox5 in Tca8113 cells by RNA-immunoprecipitation (RIP). Lysate from
Tca8113 cells were immunoprecipitated by anti-HuR antibody or IgG
antibody, and the lnc-Sox5 mRNA was validated by qRT-PCR. According to
the RT-qPCR results, lnc-Sox5 level in anti-HuR immunoprecipitated
lysate was significantly higher than in the IgG control group (Fig. 6a). To extensively confirm whether HuR is bound to
lnc-Sox5 in Tca8113 cells, we next overexpressed (Fig. 6b) or specifically inhibited HuR expression in
Tca8113 cells (Fig. 6, c and d). RT-qPCR data
indicated that overexpressing HuR could increase the level of lnc-Sox5,
while suppressed HuR expression could reduce the expression of lnc-Sox5
in Tca8113 cells. Therefore, these results suggest that lnc-Sox5 is
physically associated with and stabilized by HuR.

Fig. 6. HuR stabilized lnc-Sox5 mRNA in Tca8113 cells. a)
The HuR–mRNA complex was pulled down by anti-HuR antibody, and
the level of lnc-Sox5 was detected by qRT-PCR, IgG being the control.
b) lnc-Sox5 mRNA level was detected in Tca8113 cells when HuR was
upregulated by transfecting with HuR-expressing plasmid (CMV-HuR). c)
HuR was inhibited by transfecting with siRNA, and the inhibitory
efficacy was determined by Western blot assay. d) Expression of
lnc-Sox5 was detected by qRT-PCR in Tca8113 cells with HuR siRNAs
transfection. siNC represents the random RNA sequence and acts as the
negative control. Data are shown as mean ± S.E.M.; *** p
< 0.001.

DISCUSSION

LncRNAs have been shown to play a critical role in multiple biological
progressions including tumorigenesis [12, 13]. To date, several lncRNAs have been reported to
act as oncogene or suppressor of tongue cancer. LncRNA HOTTIP is
located at the 5′ tip of the HOXA locus and coordinates the
activation of multiple 5′ HOXA genes, which plays an important
role in multiple cancers [14]. Zhang reported that
lncRNA HOTTIP is correlated with progression and prognosis in tongue
carcinoma [9]. Additionally, it has been reported
that lncRNA UCA1 is ultra-highly expressed in tongue squamous cell
carcinoma and is correlated with tongue cancer metastasis [11]. Jia showed that miR-26a and lncRNA MEG3
expression were both strongly reduced in TSCC, and combined low
expression levels of both miR-26a and MEG3 emerged as an independent
prognostic factor for poor clinical outcome in TSCC patients [10]. In this study, we first investigated the role of
lnc-Sox5 in human tongue cancer. We observed that lnc-Sox5 expression
was upregulated in tumor tissues from 22 tongue cancer patients. To
explore whether it acted as an oncogene or a suppressor in human tongue
cancer, the lnc-Sox5 level was specifically suppressed by siRNA
transfection in tongue cancer cells. We observed lnc-Sox5 knockdown
significantly suppressed the growth of Tca8113 cells. Moreover, both
cell apoptosis and cell cycle were impaired by lnc-Sox5 knockdown.
Consistently, we also observed the invasion and migration of Tca8113
cells were suppressed by lnc-Sox5 knockdown. These results suggest that
lnc-Sox5 is an oncogene in human tongue cancer. We are still studying
the targets of lnc-Sox5 in tongue cancer cells.

HuR is a member of the ELAV (embryonic lethal abnormal vision) family of
RNA-binding proteins (RBPs), and it binds or stabilizes AU-rich element
(ARE)-containing mRNAs [15]. An ARE is commonly
present in the untranslated region of many protooncogenes, growth
factors, and cytokine mRNAs [16, 17]. Multiple copies of the sequence AU often exist
in the ARE and they target ARE-mRNAs for rapid degradation [16]. However, numerous proteins, including HuR, are
known to interact with AREs and modulate either the stabilization or
destabilization of ARE-mRNAs [18]. To date, it has
been reported that HuR also could bind to and stabilize or destabilize
lncRNA mRNAs that contained AU-rich elements [19-21]. In this study, we
observed there were many AU-rich fragments in lnc-Sox5 mRNA. RNA
immunoprecipitation suggested that HuR directly interacted with
lnc-Sox5 mRNA.

In this study, we identified very highly expressing lncRNA, lnc-Sox5, in
tumor tissues from 22 tongue cancer patients. To investigate whether
this abnormally expressing lncRNA regulated tongue cancer progression,
the expression of lnc-Sox5 was first suppressed by siRNA. MTT assays
showed that the growth of Tca8113 was dramatically suppressed by
lnc-Sox5 knockdown. Additionally, lnc-Sox5 knockdown also induced more
apoptotic Tca8113 cells. Furthermore, the cell cycle was also arrested
at G1 to S phase in Tca8113 cells. To test whether lnc-Sox5 affects
tongue cancer metastasis, we also determined the invasion and migration
in lnc-Sox5 suppressed Tca8113 cells. Transwell assays indicated that
both migration and invasion of Tca8113 cells were suppressed by
lnc-Sox5 knockdown. A xenograft tumor model suggested lnc-Sox5 promoted
tongue carcinogenesis. We also observed that there were several HuR
binding sites in lnc-Sox5 mRNA. RNA immunoprecipitation showed that HuR
directly bound to and therefore stabilized lnc-Sox5 mRNA. The direct
targets of lnc-Sox5 in Tca8113 cells are still under study. These
results suggest that lnc-Sox5, which was stabilized by HuR, may serve
as a prediction target for tongue carcinoma therapies.

Acknowledgements

This work was supported in part by grants from the Medicine and
Healthcare Development Plan in Science and Technology of Shandong
Province (Project No. 2015WSB34005 to X. Z. Ma).